DE69623485T2 - INHIBITATION OF EXOPROTEIN IN ABSORBENT PRODUCTS - Google Patents

Description

FIELD OF INVENTION

The present invention relates to the inhibition of exoprotein in an absorbent article such as vaginal tampons and sanitary napkins. In particular, the invention relates to the incorporation of ether compounds, either alone or in combination with one or more amide compounds and/or amine compounds into such absorbent articles, and the effects of these compounds on gram-positive bacteria.

BACKGROUND OF THE INVENTION

Disposable absorbent devices for absorbing human waste are widely used. These disposable devices typically comprise a compressed absorbent mass which is formed into the desired shape, which is typically dictated by the consumer's intended use. In the field of a menstrual tampon, the device is intended to be inserted into a body cavity to absorb the body fluids generally discharged during a woman's menstrual period.

There is a complex process in the female body that maintains the vagina and physiologically related areas in a healthy state. In a woman between the ages of menarche and menopause, normal Vagina provides an ecosystem for a variety of microorganisms. Bacteria are the predominant type of microorganism present in the vagina; most women have about 109 bacteria per gram of vaginal discharge. The bacterial flora of the vagina includes both aerobic and anaerobic bacteria. Bacteria more commonly isolated include Lactobacillus species, Corynebacteria, Gardnerella vaginalis, Staphylococcus species, Peptococcus species, aerobic and anaerobic Streptococcus species, and Bacteroides species. Other microorganisms occasionally isolated from the vagina include yeast (Candida albicans), protozoa (Trichomonas vaginalis), mycoplasma (Mycoplasma hominis), chlamydia (Chlamydia trachomatis), and viruses (Herpes simplex). The latter organisms are generally associated with vaginitis or sexually transmitted disease, although they may be present in lower numbers without causing symptoms.

Physiological, social and idiosyncratic factors influence the number and species of bacteria present in the vagina. Physiological factors include age, days of the menstrual cycle and pregnancy. For example, the vaginal flora present in the vagina throughout the menstrual cycle may include Lactobacilli, Corynebacterium, Ureaplasma and Mycoplasma. Social and idiosyncratic factors include birth control methods, sexual practices, systemic diseases (e.g. diabetes) and medications.

Bacterial proteins and metabolic products produced in the vagina can affect other microorganisms and the human host. For example, between menstrual periods, the vagina is slightly acidic with a pH range of about 3.8 to about 4.5. This pH range is generally considered to be the most favorable condition for maintaining normal flora. At this pH, the vagina normally hosts numerous species of microorganisms in an ecological balance, which plays a beneficial role in providing protection and resistance to infection, and which makes the vagina inaccessible to some bacterial species, such as Staphylococcus aureus (S. aureus). The low pH is a consequence of the growth of lactobacilli and their production of acidic products. Microorganisms in the vagina can also produce antimicrobial compounds, such as hydrogen peroxide, and bactericides directed against other bacterial species. An example is lactocins, bacteriocin-like products of lactobacilli, which are directed against other species of lactobacilli.

Some microbial products can affect the human host. For example, S. aureus can produce and release into its environment a variety of exoproteins, including enterotoxins, toxic shock syndrome toxin-1 (TSST-1), and enzymes such as proteases and lipases.

S. aureus is found in the vagina of approximately 16% of healthy women of menstruating age. Approximately 25% of S. aureus isolated from the vagina is capable of producing TSST-1. TSST-1 and some of the staphylococcal enterotoxins have been found to cause toxic shock syndrome (TSS) in humans.

Symptoms of TSS generally include fever, diarrhea, vomiting, and a rash followed by a rapid drop in blood pressure. Systemic failure of vital organs occurs in approximately 6% of those who come into contact with the disease. S. aureus does not cause TSS as a result of the microorganisms entering the vaginal cavity. Instead, S. aureus can cause infection through its Growing and multiplying produce toxic shock syndrome toxin-1 (TSST-1; synonyms: pyrogenic exotoxin C and enterotoxin F). Only after entering the bloodstream does TSST-1 react systemically and produce the symptoms attributed to toxic shock syndrome.

Menstrual fluid has a pH of approximately 7.3. During menstruation, the pH of the vagina changes toward neutral and may become slightly alkaline. This change allows microorganisms whose growth is inhibited by an acidic environment to proliferate rapidly. For example, S. aureus is more frequently isolated from vaginal swabs collected during menstruation than from swabs collected between menstrual periods.

Numerous attempts have been made to reduce or eliminate pathogenic microorganisms and menstrual TSS by incorporating one or more biostatic, biocidal and/or detoxifying compounds into a tampon pad. For example, L-ascorbic acid has been used in a menstrual tampon to detoxify toxin found in the woman's vagina during menstruation.

The incorporation of glycerol triacetate into a tampon pad has been proposed. Glycerol triacetate is easily degraded to glycerol and acetic acid by the enzyme action of esterase. Esterase is present in the epithelium of the vagina and in menstrual fluid. The enzyme action of esterase is in turn controlled by the pH of the environment, being more active when the pH is on the alkaline side. As the pH of the vagina moves to the alkaline side during menstruation, the enzyme action of esterase automatically increases and attacks the glycerol triacetate. This rapidly releases acetic acid, which is capable of lowering the pH and enzyme action of esterase. However, Menstrual fluid is well buffered and acetic acid is ineffective in lowering the pH of the menstrual fluid.

Others have incorporated monoesters and diesters of polyhydric aliphatic alcohols and a fatty acid of 8 to 18 carbon atoms. For example, glycerol monolaurate (GML) has been used to inhibit the production of S. aureus enterotoxins and TSST-1. However, as mentioned above, esterase is present in abundance in the epithelium of the vagina and in menstrual fluid. This esterase, in combination with esterase and lipase produced by bacteria, can enzymatically degrade the esters into inactive compounds (see, for example, EP-A-483812).

Until now, the skilled artisan has not considered the effects of lipase and esterase on ester compounds. Consequently, one or more ester compounds would have to be added to the absorbent article, such as a tampon pad, in such high concentrations that the normal flora present in the vaginal area is adversely affected. When the natural state is altered, overgrowth of pathogenic microbes may set in, leading to a condition known as vaginitis.

Accordingly, there is a need for an absorbent product having incorporated therein a compound that: effectively inhibits the production of exoproteins, such as TSST-1, from Gram-positive bacteria; is substantially unaffected by the enzymes lipase and esterase; and does not significantly alter the natural flora found in the vaginal area.

SUMMARY OF THE INVENTION

In summary, the present invention is based on the discovery that the production of exoprotein from gram-positive bacteria is substantially inhibited when an effective amount of one or more ether compounds, either alone or in combination with nitrogen-containing compounds such as amine and amide compositions, is incorporated into an absorbent article such as a menstrual tampon.

Ether compounds according to the invention are represented by the general formula:

R₁-O-R₂

wherein R₁ is a straight chain or branched alkyl group having 8 to 18 carbon atoms, and R₂ is selected from an alcohol, a polyalkoxylated sulfate salt, or a polyalkoxylated sulfosuccinate salt. It has been observed that the production of exoprotein in gram-positive bacteria is substantially inhibited when these ether compounds are incorporated into an absorbent article, such as a menstrual tampon.

Amine compounds which can be used in combination with the ether compounds according to the invention can be represented by the general formula:

wherein R₃ is an alkyl group having 8 to 18 carbon atoms; and R₄ and R₅ may be the same or different and are independently selected from Hydrogen and alkyl group(s) having 1 to 18 carbon atoms. The alkyl groups of R₄ and R₅ may contain one or more substitution moieties selected from hydroxyl, carboxyl and carboxyl salts and imidazoline. R₃ and R₄ may also form an unsaturated heterocyclic ring containing a nitrogen linked to the alpha carbon of the R₅ moiety via a double bond, thus forming a substituted imidazoline. It is also within the scope of the invention that the nitrogen-containing compound comprises an amine salt. Amine compounds and their salts have been observed to be effective in substantially inhibiting the production of exoprotein from gram-positive bacteria. Amide compositions which may be used in combination with the ether compounds of the invention may be represented by the general formula:

wherein R₇ is an alkyl group having 8 to 18 carbon atoms including the carbonyl carbon, and R₈ and R₃ may be the same or different. R₈ and R₃ are selected from hydrogen and an alkyl group having 1 to 12 carbon atoms. The alkyl group of R₈ and R₃ may comprise one or more substituent groups selected from ester, ether, amine, hydroxyl, carboxyl, carboxyl salts, sulfonate and sulfonate salts.

It is a general object of the invention to provide an absorbent article which inhibits the production of exoprotein from gram-positive bacteria.

A particular object of the invention is to provide a menstrual tampon having incorporated therein one or more ether, amide or amine compounds, either alone or in combination, which act to substantially inhibit the production of TSST-1 and enterotoxin B by S. aureus.

A further object of the invention is to provide a menstrual tampon incorporating one or more ether compounds, either alone or in combination with one or more amine or amide compounds, either alone or in combination, which substantially inhibit the production of exoproteins from gram-positive bacteria without significantly disturbing the balance of the natural flora present in the vaginal tract.

Another object of the invention is to provide a method for inhibiting the production of exoprotein produced by gram-positive bacteria in an absorbent article.

Further objects and advantages of the invention and modifications thereof will become apparent to those skilled in the art without departing from the inventive concepts defined in the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is described in detail in connection with a menstrual tampon, but would be understood by those skilled in the art to be applicable to other disposable absorbent articles such as sanitary napkins, panty liners, adult incontinence undergarments, diapers, medical dressings and tampons such as those intended for medical, dental, surgical and/or nasal use, where inhibition of exoproteins from gram-positive bacteria would be useful. Vaginal tampons suitable for use in this invention are usually made from absorbent fibers comprising natural and synthetic fibers which are compressed into a unitary body of a size which can be easily inserted into the vaginal cavity. They are normally made in an elongated cylindrical shape so as to have a sufficiently large body of material to provide the required absorbency, but can be made in a variety of shapes. The tampon may or may not be compressed, although compressed types are now generally preferred. The tampon may be made from various fiber blends comprising both absorbent and non-absorbent fibers which may or may not have a suitable sheath or coating.

It has been found that certain ether compounds can significantly inhibit the production of exoprotein from gram-positive bacteria, in particular the production of TSST-1 and enterotoxin B from S. aureus bacteria. The ether compounds of the invention have the general formula:

R₁-O-R₂

wherein R₁ is a straight or branched alkyl group having a chain of 8 to 18 carbon atoms, and R₂ is selected from an alcohol, a polyalkoxylated sulfate salt or a polyalkoxylated sulfosuccinate salt.

The alkyl or R₁ group of the ether compounds used here can be derived from saturated and unsaturated fatty acid compounds. Suitable compounds comprise C8-C18 fatty acids, and preferably the fatty acids comprise, without limitation, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid and stearic acid, whose carbon chain lengths are 8, 10, 12, 14, 16, and 18, respectively. Highly preferred materials include capric, lauric and myristic.

Preferred unsaturated fatty acids are those having one or two cis-type double bonds and mixtures of these materials. Suitable materials include myristolein, palmitolein, linolenic acid and mixtures thereof.

The R2 moiety is desirably an aliphatic alcohol which may be ethoxylated or propoxylated for use in the ether compositions of the invention. Suitable aliphatic alcohols include glycerol, sucrose, glucose, sorbitol, sorbitan and derivatives thereof. Preferred ethoxylated and propoxylated alcohols include glycols such as ethylene glycol, propylene glycol, polyethylene glycol and polypropylene glycol.

The aliphatic alcohols can be ethoxylated or propoxylated by conventional ethoxylation or propoxylation compounds and techniques. The compounds are preferably selected from the group consisting of ethylene oxide, propylene oxide and mixtures thereof, and similar ring compounds which provide a material which is effective. Most preferably, the ethoxylation compound is selected from the group consisting of ethylene oxide, propylene oxide and mixtures thereof.

The R2 moiety may further comprise polyalkoxylated sulfate and polyalkoxylated sulfosuccinate salts. The salts may have one or more cations. Preferably, the cations are sodium, potassium or both.

Preferred ether compounds of the invention include laureth-3, laureth-4, laureth-5, PPG-5 lauryl ether, 1-O- dodecyl-rac-glycerol, sodium laureth sulfate, potassium laureth sulfate, disodium laureth(3) sulfosuccinate, dipotassium laureth(3) sulfosuccinate and polyethylene oxide(2) sorbitol ether.

According to the invention, the tampon contains an effective amount of the inhibiting ether compound to substantially inhibit the formation of TSST-1 when the tampon is exposed to S. aureus bacteria. At least about 0.005 millimoles of the ether compound per gram of absorbent have been found to be effective amounts. Preferably, the ether compound is present in an amount of from 0.005 millimoles per gram of absorbent to 2 millimoles per gram of absorbent, and more preferably from 0.005 millimoles per gram of absorbent to 0.2 millimoles per gram of absorbent. Although "compound" is used in the singular, one of skill in the art would understand that it includes the plural. That is, the absorbent article can include more than one ether compound.

In another embodiment, the ether compounds of the invention can be combined with certain nitrogen-containing compounds to substantially inhibit the production of exoprotein from gram-positive bacteria, and in particular the production of TSST-1 and enterotoxin B from S. aureus bacteria. In particular, nitrogen-containing compounds are amines and their salts, having the general formula:

wherein R₃ is an alkyl group having 8 to 18 carbon atoms; and R₄ and R₅ may be the same or different and are selected from hydrogen and alkyl group(s) having 1 to 18 carbon atoms. The R₄ and R₅ alkyl groups may comprise one or more substitution moieties selected from hydroxyl, carboxyl and carboxyl salts and imidazoline. The amine compounds are effective to substantially inhibit the production of exoprotein from gram-positive bacteria.

It is significant that the R3 moiety has an alkyl group having 8 to 18 carbon atoms. The alkyl group is desirably derived from fatty acid compounds, which include, without limitation, caprylic, capric, lauric, myristic, palmitic and stearic acids, whose carbon chain lengths are 8, 10, 12, 14, 16, and 18, respectively. Highly preferred materials include capric, lauric and myristic. Preferred unsaturated fatty acids are those having one or two cis-type double bonds and mixtures of these materials. Suitable materials include myristolein, palmitolein, linolenic and mixtures thereof.

The R4 and R5 alkyl groups may further comprise one or more substitution moieties selected from hydroxyl, carboxyl, carboxyl salts, and R3 and R4 may form an unsaturated heterocyclic ring containing a nitrogen linked to the alpha carbon of the R5 moiety by a double bond, thus forming a substituted imidazoline. The carboxyl salts may have one or more cations selected from sodium, potassium, or both. The R3-R5 alkyl groups may be straight chain or branched, and may be saturated or unsaturated.

In another respect, the amine compound can be a salt. The salt can be represented by the general formula:

where R3 is an anionic moiety attached to the amine and derived from an alkyl group having 8 to 18 carbon atoms. R3 is desirably a polyalkyloxylated alkyl sulfate. The R6 moiety is the same as described above for the R4 and R5 moieties. The R3-R6 alkyl groups may be straight chain or branched, and may be saturated or unsaturated. A preferred compound exemplifying an amine salt is TEA laureth sulfate.

Preferred amine compounds which can be incorporated into the absorbent article or onto the cover thereof include TEA laureth sulfate, lauramine, lauraminopropionic acid, sodium lauriminodipropionic acid, lauryl hydroxyethylimidazoline, and mixtures thereof.

According to the invention, the tampon contains, in addition to the ether compounds of the invention, an effective amount of the inhibiting nitrogen-containing compound to substantially inhibit the formation of TSST-1 when the tampon is exposed to S. aureus bacteria.

At least 1·(10⁻⁵) millimoles of the amine compound per gram of absorbent have been found to be effective amounts. Preferably, the amine compound is present in an amount of from 0.005 millimoles per gram of absorbent to 2 millimoles per gram of absorbent, and more preferably from 0.005 millimoles per gram of absorbent to 0.2 millimoles per gram of absorbent. Although "compound" is used in the singular, one skilled in the art would understand that it includes the plural. That is, the absorbent article may comprise more than one amine compound.

In a further aspect of the invention, the nitrogen-containing compounds have the general formula:

wherein R₇ is an alkyl group having 8 to 18 carbon atoms including the carbonyl carbon, and R₈ and R₃ may be the same or different. R₈ and R₃ are selected from hydrogen and an alkyl group having 1 to 12 carbon atoms, which may include one or more substituent groups selected from ester, ether, amine, hydroxyl, carboxyl, carboxyl salts, sulfonate and sulfonate salts.

The alkyl moiety including the carbonyl carbon can be derived from saturated and unsaturated fatty acid compounds. Suitable compounds include C8-C18 fatty acids, and preferably the fatty acids include, without limitation, caprylic, capric, lauric, myristic, palmitic and stearic acids, whose carbon chain lengths are 8, 10, 12, 14, 16 and 18 respectively. Highly preferred materials include capric, lauric and myristic.

Preferred unsaturated fatty acids are those having one or two cis-type double bonds and mixtures of these materials. Suitable materials include myristolein, palmitolein, linolenic and mixtures thereof.

The R8 and R9 moieties may be the same or different and each may be selected from hydrogen and an alkyl group having a carbon chain of 1 to 12 carbon atoms. The R7 -R9 alkyl groups may be straight or branched chain and may be saturated or unsaturated. When R8 and/or R9 is an alkyl moiety having a carbon chain of at least 2 carbons, the alkyl group may comprise one or more substituent groups selected from ester, ether, amine, hydroxyl, carboxyl, carboxyl salts, sulfonate and sulfonate salts. The salts may have one or more cations selected from sodium, potassium or both.

Preferred amide compounds include sodium lauroyl sarcosinate, lauramide MEA, lauramide DEA, lauramidopropyl dimethylamine, disodium lauramido MEA sulfosuccinate, and disodium lauroamphodiacetate.

According to the invention, the tampon contains, in addition to the ether compounds of the invention, an effective amount of the inhibiting nitrogen-containing compound to substantially inhibit the formation of TSST-1 when the tampon is exposed to S. aureus bacteria. At least 5 x (10⁻⁴) millimoles of the amide compound per gram of absorbent have been found to be effective amounts. Preferably, the amide compound is present in an amount of from 0.005 millimoles per gram of absorbent to 2 millimoles per gram of absorbent. More preferably, the amide compound is present in an amount of from 0.005 millimoles per gram of absorbent to 0.2 millimoles per gram of absorbent. Although "compound" is used in the singular, one skilled in the art would understand that it includes the plural. That is, the absorbent article can include more than one amide compound. The compositions according to the invention can be prepared and applied in any suitable form, but are preferably prepared in forms comprising, without Restriction, aqueous solutions, lotions, balms, gels, ointments, unguents, pills, suppositories and the like.

The compositions can be applied to the absorbent article using conventional methods for applying an inhibiting agent to the desired absorbent article. For example, one-piece tampons without separate wrappers can be immersed directly in a bath liquid containing the agent and can then be air dried if necessary to remove any volatile solvents. For compressed tampons, soaking each of their elements is best done prior to compression. When the compositions are incorporated onto and/or into the tampon material, they can be loose, loosely glued, bonded, or any combination thereof. As used herein, the term "loose" means that the composition is capable of migrating through the tampon materials.

It is not necessary to soak the entire absorbent tampon body with the inhibiting agent. Optimum results, both economically and functionally, can be obtained by concentrating the material on or near the outer surface where it is most effective during use.

The substantially inhibiting composition may additionally employ one or more conventional pharmaceutically acceptable and compatible carrier materials suitable for the desired application. The carrier may be suitable for bringing the materials used in the composition into solution or for dispersing them together. Carrier materials suitable for use in the present composition therefore include those which are suitable for use in the cosmetic and medicinal fields as a basis for unguents, lotions, creams, ointments, aerosols, suppositories, gels and the like.

The inhibiting composition of the invention may also use additional components which are conventionally found in pharmaceutical compositions of the art and of the corresponding standard of the art. For example, the compositions may additionally comprise suitable pharmaceutically active materials for combination therapy, such as antimicrobial additives, antiparasitic agents, antipruritic agents, astringents, local anesthetics or anti-inflammatory agents.

The present invention can be readily understood by considering the following examples illustrating specific embodiments. The examples are intended to serve as an aid to carrying out the invention, and are not to be construed as a limitation or limitations on the invention. It is to be understood that numerous changes or modifications may be made as would be apparent to those skilled in the art without departing from the scope of the claims appended hereto.

EXAMPLES 1-3 (Examples 2 and 3 for comparison)

The potency of the test compounds on the production of TSST-1 was determined by adding the desired concentration, expressed in millimoles/milliliter (millimolar, hereinafter mM), of the active compound to 10 milliliters of growth medium of each test compound in a 50 ml Corning polystyrene conical tube. The polystyrene tube is available from Scientific Products Division, Baxter Diagnostics Incorporated, 1430 Waukegan Road, McGaw Park, IL 60085-6787.

Growth medium was prepared as follows: Brain Heart Infusion Broth (BHI), available from Becton Bickinson Microbiology Systems, Cockeysville, MD 21030, was reconstituted and sterilized according to manufacturing instructions. Ninety milliliters of BHI medium was supplemented with 10 ml of fetal bovine serum (FBS), available from Sigma Chemical Company, P. O. Box 14508, St. Louis, MO 63178-9916. One milliliter of a 0.002 molar sterile solution of magnesium chloride hexahydrate, available from Sigma Chemical Company, was added to the BHI-FBS mixture. One milliliter of a 0.027 molar sterile solution of L-glutamine, available from Sigma Chemical Company, was also added to the BHI-FBS mixture.

If the test compound was not water soluble or water miscible, it was first dissolved in 10 ml of isopropanol at 50-fold the desired concentration, then diluted to the desired final concentration in 10 ml of growth medium. Tubes of growth medium containing an equivalent amount of isopropanol but without test compound were prepared as controls.

In preparation for inoculating growth medium tubes with the test compound, an inoculation medium was prepared as follows. S. aureus, (MN8) was streaked onto a sheep blood agar plate and incubated at 37ºC. The test organism in this example was obtained from Dr. Pat Schlievert, Department of Microbiology of the University of Minnesota Medical School, Minneapolis, MN. After 24 hours of incubation, three to five single colonies were picked with a sterile inoculation loop and used to inoculate 10 ml of growth medium. The tube containing the inoculated growth medium was capped with an S/P®diSPo® stopper, available from Scientific Products Division, Baxter Diagnostics, Incorporated, and incubated at 37ºC under an air atmosphere containing 5% CO₂ (vol.). After 24 hours of incubation, the culture was removed from the incubator and mixed vigorously on an S/P brand vortex mixer. A second tube containing 10 ml of growth medium was inoculated with 0.5 ml of the above 24 hour culture and again incubated at 37ºC under air atmosphere with 5% CO₂ by volume. After 24 hours of incubation, the culture was removed from the incubator and mixed vigorously on an S/P brand vortex mixer. Each tube containing growth medium containing test compound, and growth control tubes with or without isopropanol, were inoculated with 0.1 ml of the prepared inoculation medium. The initial colony forming units (CFU) per ml of growth medium was approximately 1 x 10⁷. The tubes were sealed with S/P®diSPo® stoppers and incubated at 37ºC under air atmosphere with 5% CO₂ by volume. After 24 hours of incubation, the tubes were removed from the incubator and the culture fluid was analyzed for the number of colony forming units (CFU) of S. aureus and prepared for analysis for TSST-1 using the method described below.

The number of colony forming units per ml after incubation was determined by standard plate counting procedures. The culture fluid medium was centrifuged and the supernatant was then sterile filtered through an Acrodisc® syringe filter unit available from Scientific Products Division, Baxter Diagnostics, Inc. The resulting fluid was stored frozen at -80ºC until assayed.

The amount of TSST-1 per milliliter was determined by a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA). Samples of the culture fluid and the TSST-1 reference standard were analyzed three times. The The method used was as follows: Four reagents, rabbit polyclonal anti-TSST-1 IgG (#LTI-101), rabbit polyclonal anti-TSST-1 IgG conjugated to horseradish peroxidase (#LTC-101), TSST-1 (#TT-606), and normal rabbit serum guaranteed anti-TSST-1 free (#NRS-10), were purchased from Toxin Technology, Incorporated, 7165 Curtiss Avenue, Sarasota, Florida, 34231. Sixty-two microliters of rabbit polyclonal anti-TSST-1 IgG (#LTI-101) was appropriately diluted so that a 1:100 dilution gave an absorbance of 0.4 at 650 nanometers. These were added to 6.5 ml of a 0.5 molar carbonate buffer, pH 9.6, and 100 microliters of this solution was pipetted into the inner wells of polystyrene microtiter plates #439454, available from Nunc-Denmark. The plates were covered and incubated overnight at 37ºC. Unbound antitoxin was removed by washing three times with phosphate buffered saline (pH 7.2) (0.011 molar NaH2PO4 and 0.9% [w/v] NaCl, both available from Sigma Chemical Company) containing 0.5% [vol/vol] Tween 20 (PBS-Tween) (PBS, Phosphate Buffered Saline), also available from Sigma Chemical Company. Plates were treated with 100 microliters of a 1% [w/v] solution of bovine serum albumin (BSA) available from Sigma Chemical Company, covered, and incubated for one hour at 37ºC. Unbound BSA was removed by washing six times with PBS-Tween. TSST-1 reference standard serially diluted from 1-10 ng/ml in PBS-Tween, test samples treated with normal rabbit serum to 10% [vol/vol] final concentration, and reagent controls were pipetted into the appropriate wells in volumes of 100 microliters, followed by incubation for two hours at 37ºC and washing three times to remove unbound toxin. Rabbit polyclonal anti-TSST-1 IgG conjugated to horseradish peroxidase (100 microliter volumes) diluted according to the manufacturer's instructions was added to each microtiter well. The plates were covered and incubated at 37ºC for one hour. After incubation, the plates were washed 6 times in PBS-Tween. Subsequently, the wells were treated with a solution consisting of 0.075 molar sodium citrate (pH 4.0), 0.6 millimolar 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and 0.009% [vol/vol] hydrogen peroxide, all available from Sigma Chemical Company. The intensity of the color reaction in each well was evaluated as a function of time using a BioTek Model EL340 microplate reader (OD 405 nm) and Kineticalc® software available from Biotek Instruments, Inc. TSST-1 concentrations in test samples were derived from the regression reference equations for toxin during each analytical method.

The effectiveness of the ether compounds of the invention, as well as that of amine and amide compounds as comparative examples, in inhibiting the production of TSST-1 is shown below in the respective Tables I-III. Table I

ND = Not detected (CFU = Colony forming units)

The above list of compounds (commercial name), their active compound percentage, and supplier are as follows:

1-O-Dodecyl-rac-glycerol, Sigma Chemical Company, 100%, P.O. Box 14508, St. Louis, MO 63178-9916.

Laureth-3, (Trycol 5966), 97%, Henkle Corporation, Emery Group, 4900 Este Avenue, Cincinnati, Ohio, 45232.

Laureth-4, (Trycol 5882), 100%, Henkle Corporation, 300 Brookside Avenue, Ambler, Pennsylvania 19002.

Sodium laureth sulfate (Standapol ES-2), 25%, Henkle Corporation. PPG-5-Laureth-5, (Aethoxal B), 100%, Henkle Corporation.

Disodium laureth sulfosuccinate, (Standapul SH124-3), 39%, Henkle Corporation.

According to the present invention, the data in Table I show that S. aureus MN8 produced significantly less TSST-1 in the presence of the ether compounds when compared to the control. The ether compounds reduced the amount of exotoxin production by about 95 percent to greater than 99.6 percent. However, although the amount of toxin produced was significantly reduced, in most cases the ether compounds did not significantly reduce the number of S. aureus cells. Table II (comparative example)

*S. aureus grew in the culture medium, the amount of growth was not determined.

The above list of compounds (commercial name), their active compound percentage, and supplier are as follows:

Lauriminopropionic acid, (Mackam 151L), 40%, McIntyre Group, LTD, 1000 Governors Highway, University Park, IL. 60466.

Sodium lauriminodipropionic acid, (Deriphat 160-C), 30%, Henkle/Cospha, Cospha Group, 300 Brookside Ave., Ambler, PA 19002.

Laurylhydroxyethylimidazoline, (Schercozolin L), Scher Chemicals, Inc., Industrial West, P.O. Box 4317, Clifton, NJ 07012.

TEA laureth sulfate, (Sulfochem TLES), 39%, Chemron, P O Box 2299, Paso-Robles, CA 93447.

Lauramin, (dodecylamine), 99+, Aldrich, 1001 West Saint Paul Ave, Milwaukee, WI, 53233.

The data in Table II show that S. aureus MN8 produced significantly less TSST-1 in the presence of the amine compounds when compared to the control. The amine compounds reduced the amount of exotoxin production by approximately 86 percent to greater than 99.4 percent. Table III (Comparative Example)

The above list of compounds (commercial name), their active compound percentage, and supplier are as follows:

Lauramid-MEA, (Comperian LNN), 98.5%, Henkle Corporation, 300 Brookside Avenue, Ambler, Pennsylvania 19002.

Sodium lauroyl sarcosinate, (Hamposyl L-30), 30%, Hampshire Chemical Co., 55 Hayden Ave., Lexington, MA 02173.

Disodium lauroamphodiacetate, (Mackam 2-L), 50% McIntyre Group, 1000 Governors Highway, University Park, IL. 60466.

Disodium lauramido MEA sulfosuccinate, (Mackanate LM-40), 40%, McIntyre Group, 1000 Governors Highway, University Park, IL. 60466.

The data in Table III show that S. aureus MN8, when compared to the control, produced significantly less TSST-1 in the presence of the amide compounds. The amide compounds reduced the amount of exotoxin production by approximately 86 percent to greater than 99.6 percent.

EXAMPLES 4-6 (Examples 5 and 6 for comparison)

The effectiveness of the test compounds in reducing the production of a second exoprotein from S. aureus was determined using S. aureus HOCH, a known producer of enterotoxin B. The test organism in this example was obtained from Dr. Pat Schlievert, Department of Microbiology of the University of Minnesota Medical School, Minneapolis, MN. The experimental procedure to demonstrate the effectiveness of the test compound action on the growth of S. aureus HOCH and to produce a culture fluid was the same as shown above in Example A.

The amount of S. aureus enterotoxin B per milliliter was determined by Western blot analysis. Culture fluid samples and the reference standard for staphylococcal enterotoxin B (SEB) were analyzed in triplicate. The method used was similar to that described in "A Rapid Sensitive Method for Detection of Alkaline Phosphatase-Conjugated Anti-Antibody on Western Blots," MS Blake, KH Johnston, GJ Russell-Jones, and EC Gotschlich, Analytical Biochemistry, 136: 175-179, 1984. Enterotoxin B was separated from other proteins in the test samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 3% acrylamide gel was used for the upper stacking gel, and a 14% acrylamide gel was used for the lower separating gel. The top gel was prepared with a comb containing 20 lanes spanning 15.5 centimeters of the top of the stacking gel. A low molecular weight SDS-PAGE standard, BioRad#161-0305, available from Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547, SEB, #BT-202 from Toxin Technology, Incorporated, and culture extracts prepared as above were each mixed 1:1 with a loading buffer. The loading buffer contained 30% [vol/vol] glycerol, 15% [vol/vol] mercaptoethanol, 7% [wt/vol] sodium dodecyl sulfate, 0.0036% [wt/vol] bromophenol blue, and 0.76% [wt/vol] Trizma base at pH 6.8. The mixtures were boiled for 5 minutes, after which twenty-five (25) microliters of each mixture was added to a lane. The SDS-PAGE gel was electrophoresed (60 to 80 volts) for 90 minutes or until the dye front had passed through the stacking gel, and for an additional 2.5 hours (at 160-170 volts) with an electrophoresis buffer consisting of 0.61% [wt/vol] Tris base, 2.85% [wt/vol] glycine, 0.1% [wt/vol] SDS, pH 7.85. Proteins were electrophoresed overnight for approximately 15 hours at 200 milliamperes. in a Bio-Rad Trans-Blot® cell onto a nitrocellulose transfer membrane available from Schleicher and Schüll, Inc., Keene, NH 03431, #BA 85. The transfer buffer was composed of 0.3% [wt/vol] Tris base, 1.4% [wt/vol] glycine, 20% [vol/vol] methanol, pH 7.6.

The nitrocellulose membrane was treated with 3% [w/v] gelatin in 0.02 molar Tris buffer, 0.5 molar NaCl, pH 7.5 (TBS) for 45 minutes at 37ºC to inhibit nonspecific reactions, then washed with TBS-0.05% [vol/vol] Tween 20 (TBS-Tween) for 45 minutes at 37ºC. The membrane was then immersed in 50 ml of TBS-Tween containing 0.05% rabbit polyclonal anti-SEB IgG, #LBI-202, available from Toxin Technology, Incorporated, for 1.5 hours at 37ºC.

The membrane was washed twice in TBS-Tween, then immersed a second time for 1.5 hours at 37ºC in a 50 ml solution of TBS-Tween containing 25 microliters of goat anti-rabbit IgG conjugated to alkaline phosphatase. The membrane was washed twice in TBS-Tween and twice in TBS. The blot was developed with a reaction solution consisting of 2 mg of 5-bromo-4-chloroindolyl phosphatase, 100 microliters of N,N-dimethylformamide, 18 ml of 0.15 molar barbitol buffer, pH 9.2, 2 mg of nitroblue tetrazolium, and 40 microliters of a 2 molar solution of MgCl2·6H2O, all available from Sigma. The reaction was stopped by washing with cold water. The amount of SEB produced in the presence of the test compound was estimated by comparison with the color intensity obtained with a series of dilutions of SEB.

The effectiveness of the ether compounds according to the invention, as well as that of the amine and amide compounds with regard to The inhibition of the production of enterotoxin B is shown below in the respective Tables IV-VI. Table IV

ND = Not detected, < 0.16 mg/ml

According to the present invention, the data in Table IV show that S. aureus HIGH, when compared to the control, produced significantly less SEB in the presence of Although the amount of toxin produced was significantly reduced, the ether compounds did not significantly reduce the number of S. aureus cells in most cases. Table V (Comparative example)

*S. aureus grew in the culture medium, the amount of growth was not determined.

The data in Table V show that S. aureus HIGH produced significantly less SEB in the presence of the amine compounds. When compared to the control, the amine compounds reduced the amount of exoprotein production below the detectable range of 0.16 milligrams/milliliter. Table VI (comparative example)

*No SEB detected at a concentration of 0.43 millimoles sodium lauroyl sarcosinate, no counting on the plates was performed.

Not detected = < 0.16 mg/ml.

The data in Table VI show that S. aureus HIGH produced significantly less SEB in the presence of the amide compounds when compared to the control. Although the amount of toxin produced was reduced to below the detection limit, the concentration of the amide compound can be adjusted to result in only a small reduction in the number of S. aureus cells. The amide compounds reduced the amount of exotoxin production to below the detectable range of 0.16 milligrams/milliliter.

Another aspect of the invention is a method for inhibiting the production of exoprotein from gram-positive bacteria in an absorbent product. The method comprises the steps of contacting the absorbent article, such as a tampon, with one or more of the inhibiting compositions described above, thereafter applying the absorbent article to use so that the production of exoprotein from gram-positive bacteria is inhibited. The absorbent article may have one or more inhibiting compositions absorbed into or applied to the fiber or cover material. Desirably, the production of TSST-1 and enterotoxin B is inhibited.

Although the invention has been described in connection with a particular embodiment, it is understood that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, the invention is intended to embrace all alternatives, modifications and variations which fall within the scope of the appended claims.

Claims (17)

1. An absorbent article comprising an effective amount of one or more ether compounds having the general formula: R₁-O-R₂ wherein R₁ is a straight or branched, saturated or unsaturated alkyl group having a chain of 8 to 18 carbon atoms, and R₂ is selected from an alcohol, a polyalkoxylated sulfate salt, and a polyalkoxylated sulfosuccinate salt, wherein the compound is effective to substantially inhibit the production of exoprotein from gram-positive bacteria. 2. Absorbent article according to claim 1, wherein the alkyl group is derived from caprylic, capric, lauric, myristic, palmitic or stearic acid. 3. An absorbent article according to claim 1 or 2, wherein the alcohol is an aliphatic alcohol. 4. The absorbent article of claim 3, wherein the aliphatic alcohol is selected from glycerol, glycol, sucrose, glucose, sorbitol, sorbitan and derivatives thereof. 5. The absorbent article of claim 4, wherein the glycol is selected from ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol. 6. The absorbent article of claim 1 or 2, wherein the cationic moiety of the sulfate salt and the sulfosuccinate salt is selected from sodium, potassium, or both. 7. The absorbent article of claim 1 or 2, wherein the compound is selected from laureth-3, laureth-4, laureth-5, PPG-5 lauryl ether, 1-O-dodecylrac-glycerol, sodium laureth sulfate, potassium laureth sulfate, disodium laureth(3) sulfosuccinate, dipotassium laureth(3) sulfosuccinate and polyethylene oxide(2) sorbitol ether and combinations thereof. 8. An absorbent article according to any one of the preceding claims, wherein the compound is present in an amount greater than 0.005 millimoles per gram of absorbent. 9. The absorbent article of claim 8, wherein the compound is present in an amount greater than 0.005 millimoles per gram of absorbent to 2 millimoles per gram of absorbent. 10. The absorbent article of claim 1, wherein R₁ is a straight or branched, saturated or unsaturated alkyl group having a chain of 8 to 18 carbon atoms derived from caprylic, capric, lauric, myristic, palmitic and stearic acid, and R₂ is selected from an aliphatic alcohol, polyalkoxylated sulfate salt and polyalkoxylated sulfosuccinate salt, wherein the aliphatic alcohol is selected from glycerol, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, Sucrose, glucose, sorbitol, sorbitan and derivatives thereof, wherein the compound is effective to substantially inhibit the production of TSST-1 and enterotoxin B from S. aureus bacteria. 11. The absorbent article of claim 10, wherein the cationic moiety of the sulfate salt and the sulfosuccinate salt is selected from sodium, potassium, or both. 12. The absorbent article of claim 10, wherein the compound is selected from laureth-3, laureth-4, laureth-5, PPG-5 lauryl ether, 1-O-dodecyl-rac-glycerol, sodium laureth sulfate, potassium laureth sulfate, disodium laureth(3) sulfosuccinate, dipotassium laureth(3) sulfosuccinate and polyethylene oxide(2) sorbitol ether. 13. An absorbent article according to any one of the preceding claims, wherein the one or more ether compounds are in combination with an effective amount of one or more nitrogen-containing compounds having the general formula: wherein R₃ is a straight-chain or branched, saturated or unsaturated alkyl group having 8 to 18 carbon atoms; and R₄ and R₅ may be the same or different and are selected from hydrogen and a straight-chain or branched, saturated or unsaturated alkyl group having 1 to 18 carbon atoms, and which may have one or more substitution groups selected from hydroxyl, carboxyl and carboxyl salts and imidazoline, or wherein R₃ and R₄ may form an unsaturated heterocyclic ring containing a nitrogen linked by a double bond to the alpha carbon of the R₅ moiety to form a substituted imidazoline, said compound being effective to substantially inhibit the production of exoprotein from gram-positive bacteria and or an effective amount of one or more nitrogen-containing compounds having the general formula: wherein R₇ is a straight chain or branched, saturated or unsaturated alkyl group having 8 to 18 carbon atoms, including the carbonyl carbon, and R₈ and R₉ may be the same or different, wherein R₈ and R₉ are selected from hydrogen, a straight chain or branched, saturated or unsaturated alkyl group having 1 to 12 carbon atoms, and may contain one or more substituent groups selected from ester, ether, amine, hydroxyl, carboxyl, carboxyl salts, sulfonate, sulfonate salts, and combinations thereof, wherein the compound is effective to substantially inhibit the production of exoprotein from gram-positive bacteria. 14. A method for inhibiting the production of exoprotein from Gram-positive bacteria in a absorbent product comprising contacting the absorbent product with an effective amount of one or more ether compounds and exposing the absorbent product to one or more gram-positive bacteria, wherein the ether compound has the general formula: R₁-O-R₂ wherein R₁ is a straight-chain or branched, saturated or unsaturated alkyl group having a chain of 8 to 18 carbon atoms, and R₂ is selected from an alcohol, a polyalkoxylated sulfate salt and a polyalkoxylated sulfosuccinate salt. 15. The method of claim 14, wherein the ether compound is selected from laureth-3, laureth-4, laureth-5, PPG-5 lauryl ether, 1-O-dodecyl-rac-glycerol, sodium laureth sulfate, potassium laureth sulfate, disodium laureth(3) sulfosuccinate, dipotassium laureth(3) sulfosuccinate and polyethylene oxide(2) sorbitol ether. 16. The method of claim 14 or 15, wherein the absorbent product is a menstrual tampon. 17. A method according to any one of claims 14 to 16, wherein the absorbent product is additionally treated with an effective amount of one or more nitrogen-containing compounds having the general formula: wherein R₃ is a straight-chain or branched, saturated or unsaturated alkyl group having from 8 to 18 carbon atoms; and R₄ and R₅ may be the same or different and are selected from hydrogen and a straight-chain or branched, saturated or unsaturated alkyl group having from 1 to 18 carbon atoms, and which may have one or more substitution moieties selected from hydroxyl, carboxyl and carboxyl salts and imidazoline, or wherein R₃ and R₄ are can form an unsaturated heterocyclic ring containing a nitrogen linked to the alpha carbon of the R5 moiety via a double bond, thus forming a substituted imidazoline, the compound being effective to substantially inhibit the production of exoprotein from gram-positive bacteria and or an effective amount of one or more nitrogen-containing compounds having the general formula: wherein R₇ is a straight-chain or branched, saturated or unsaturated alkyl group having 8 to 18 carbon atoms, including the carbonyl carbon, and R₈ and R₆ may be the same or different, wherein R₈ and R₆ are selected from hydrogen, a straight-chain or branched, saturated or unsaturated alkyl group having 1 to 12 carbon atoms, and may contain one or more substituent groups selected from ester, ether, amine, hydroxyl, carboxyl, carboxyl salts, sulfonate, sulfonate salts, and combinations thereof, wherein the compound is effective to substantially inhibit the production of exoprotein from gram-positive bacteria.